KPC-2碳青霉烯酶对高毒力肺炎克雷伯菌荚膜多糖CPS表达的调控

Hypervirulent Klebsiella pneumoniae(hvKP) showed high virulence and they have good sensitivity to frequently-used antibiotics. However, carbapenem-resistant hvKP (CR-hvKP) which mainly produced KPC-2 type carbapenemase has appeared in China. Our previous study found that compared with hvKP, the total pathogenicity of CR-hvKP decreased and the frequency or expression of genes encoding capsular polysaccharide (CPS) synthesis significantly decreased in CR-hvKP, which indicated that the acquisition of blakpc-2 in hvKP may down-regulate CPS directly or indirectly, and then reduce the virulence of bacteria, but the regulatory mechanism remains unclear. We will construct CR-hvKP by in vitro overexpression of blakpc-2 then transform into hvKP to clarify the mechanism that how blaKPC-2 regulates CPS expression in the same genetic background from gene and transcriptional level, in vitro and in vivo experiments. We will study the possible regulatory mechanism of blaKPC-2 on CPS expression using RNA-SEQ, gene knockout and complementation and electrophoretic mobility shift assay (EMSA). The findings will provide ideas for analysing the fitness association between resistance and virulence and provide drug target for new anti-infective drugs.

高毒力肺炎克雷伯菌(hvKP)致病力强，对临床常用抗菌药物敏感性较好，但国内已出现碳青霉烯类耐药的hvKP(CR-hvKP)，主要产KPC-2碳青霉烯酶(blaKPC-2)。申请人前期发现CR-hvKP较hvKP临床株相比总体致病力有所降低，重要毒力分子荚膜多糖(CPS)合成相关基因在CR-hvKP临床株携带率或表达较hvKP明显降低，提示hvKP获得blakpc-2后通过直接或间接方式下调CPS的表达，进而降低细菌毒力，但具体调控机制不清。本项目拟采用体外过表达blakpc-2并转化hvKP方法构建CR-hvKP，从转录和蛋白水平、从体外和体内实验明确相同遗传背景下blaKPC-2对hvKP CPS表达的影响。通过RNA-SEQ、基因敲除及回补、凝胶迁移实验等方法分析blaKPC-2对CPS表达调控机制。研究结果为解析细菌致病力与耐药性的适应性关系、为设计新的抗感染药物提供依据和靶点。

目的：课题组前期发现携带blaKPC-2的CR-hvKP临床株较hvKP相比总体致病力有所降低，重要毒力分子荚膜多糖(CPS)合成相关基因及调控基因在CR-hvKP临床株携带率或表达较hvKP明显降低，提示hvKP获得blaKPC-2后通过直接或间接方式下调CPS表达，进而降低细菌毒力，但具体调控机制不清。本项目拟明确相同遗传背景下blaKPC-2对hvKP CPS表达的影响。.内容：收集本院近几年临床分离的肺炎克雷伯菌对其进行分子流行病学分析。选取3株有代表性的hvKP通过体外过表达blaKPC-2并转化hvKP的方法得到CR-hvKP(即blaKPC-2转化株)。采用药敏试验检测hvKP野生株和blaKPC-2转化株对碳青霉烯类药物的敏感性；采用黏液拉丝实验、血清抵抗实验、生物膜形成实验比较hvKP野生株和blaKPC-2转化株毒力表型差异；沉降实验和苯酚-硫酸法检测细菌黏液性及糖醛酸含量；RT-PCR方法检测hvKP野生株和blaKPC-2转化株CPS结构基因及相关调控基因表达水平。.结果：分子流行病学研究显示，CRKP耐药率高但毒性低，以ST11为主，hvKP毒力高但耐药率低，以ST23和ST65为主，血清型以K1型和K2型为主。课题组成功将blaKPC-2克隆至质粒pHSG396并转化至hvKP临床株，得到blaKPC-2转化株。与hvKP相比，blaKPC-2转化株对碳青霉烯类药物转为耐药，黏液拉丝实验转为阴性，对血清中杀菌物质的抵抗能力明显降低，生物膜形成能力降低，细菌黏液性下降，糖醛酸含量明显降低。RT-PCR结果显示，与hvKP相比，blaKPC-2转化株CPS结构基因orf1(galF)、orf3(wzi)、orf16(manC)及粘液相关基因rmpA、rmpA2表达下调。以上实验均表明，blaKPC-2可通过降低hvKP CPS的表达，进而降低细菌毒力。.结论与研究意义：本研究表明，blaKPC-2可通过降低CPS的表达，导致细菌毒力降低。该研究可为临床抗感染治疗提供理论依据，为新的抗菌药物的设计提供靶标。